PROJECT SUMMARY Spinal cord injury (SCI) has no effective treatments in the clinic. More recently, stem cell therapy has yielded encouraging early results in SCI repair. However, cell-based therapies face numerous challenges such as immune incompatibility, cell survival, and long-term safety. Soluble factors secreted by the cells (secretome) are believed to be key players in the effectiveness of cell therapy, and present a number of advantages over the use of cells in terms of safety, manufacturing, storage, and handling as a ready-to-go biologic product. In the proposed project, we plan to use secretome from adipose derived stromal/stem cells (ASCs) because ASCs secrete neurotrophic factors (e.g., BDNF, NGF) and are effective for neural regeneration in vivo, as demonstrated by Co-I Dr. March. Moreover, ASCs are clinically relevant because they are easily accessible and in abundant supply. Additionally, electrical stimulation (ES) is used as a non-drug approach for promoting cell migration within the central nervous system for restoration and repair after neural damage. However, delivering electrical fields in a less invasive manner is a challenge, as most effective ES methods require surgical implantation of electrodes at the lesion site. In this project, we propose using the secretome of electrically stimulated ASCs to provide benefits of both ES and stem cell therapy while eliminating limitations and complications traditionally associated with those techniques. Importantly, we have shown that ES can promote the production of distinct proteins in cell secretome. To define optimal parameters for ES, experimentally derived electrochemical modeling is essential. Collaborator Dr. Patrick has developed a preliminary model using equivalent circuits to characterize the electro-bioreactor used in the proposed studies. Another hurdle to SCI therapy is delivery of the therapeutics at the lesion site. One strategy is to use injectable hydrogels since they are minimally invasive and fill irregularly shaped lesions. Hydrogels derived from decellularized rat peripheral nerve were previously developed by PI Dr. Schmidt, and were shown to partially restore function after SCI in rats. Use of this hydrogel will maintain the secretome at the lesion site and provide pro-regenerative extracellular matrix proteins to enhance axonal regeneration. We propose to develop and test an injectable system for SCI repair consisting of a hydrogel derived from decellularized nerve in combination with novel secretome from electrically stimulated ASCs. In Aim 1, we will produce and characterize pro-regenerative secretome from ASCs by means of ES, as informed by electrochemical modeling. In Aim 2, we will develop injectable hydrogel from decellularized nerve and optimize it for secretome release and injection using in vitro and in vivo studies. Aims 1 and 2 are independent, and experiments from both aims will be performed in parallel for expeditious processing and analysis. Finally, success in the proposed research could lead to applications of secretome from electrically stimulated cells for tissue engineering applications to treat other diseases and injuries.